Characteristics Of Catechol 1,2-Dioxygenases Involved In Degradation Of Polycyclic Aromatic Hydrocarbons By Achromobacter Xylosoxidans DN002

Polycyclic aromatic hydrocarbons(PAHs)are a class of organic pollutants with highstability,strong-hydrophobicity and recalcitrance.That has caused serious harm to the environment and human body because of toxic,mutagenic and carcinogenic.Thence,PAHs pollution has become one of the most urgent environmental problems to be solved.Microbial remediation is considered to be the best option for the removal of PAHs,and it has been shown that oxygenase produced by bacteria can degrade PAHs effectively.Catechol 1,2-dioxygenase(CatA)is an essential enzyme in the degradation of PAHs,which mainly catalyzes the deoxidation of catechol and the ring cleavage of intradiol to promote the degradation of PAHs.Therefore,Achromobacter xylosoxidans DN002 was chosen to investigate the functional characterization of the CatA by means of molecular biological approach combined with homology modeling,molecular docking and multi-spectroscopy aiming to clarify the potential binding interaction between CatA and catechol.A gene(cat A)knockout strategy was used to construct the mutant strain of the Δ cat A,complementation strain of cat A-C and overexpression strain of DN-cat A in the DN002 for exploring their growth and degradation rate of catechol.The results showed that the removal rate of catechol was 54.21% by the wild strain DN002.However,the growth and degradation rate of mutant strain was slow due to the gene deletion under the same condition,which degradative rate only is 12.03%.In addition,the degradative rate of catechol by cat A-C was53.18%,which was basically consistent with the wild-type level,while the degradative rate of catechol by DN-cat A was as high as 88.24%.Then the expression of cat A gene and other degradation genes induced by catechol in four strains was analyzed using q RT-PCR.The results showed that the expression levels of degradation genes were regulated to varying degrees under the induction of catechol in the wild-type strain DN002.Moreover,the expression of cat C was decreased by the deletion of cat A gene,while the expression of gtd A and hmg A was increased.It can be seen that the degradation process requires a variety of enzymes to work together,and the change of cat A significantly affects the expression of other vital genes.On the basis of preliminary experiment,the protein exogenous expression and purification of CatA were carried out,and the optimal conditions of the enzyme were determined.The binding interaction between CatA and catechol was clarified with homology modeling,molecular docking and multispectral analysis.The three-dimensional structure of CatA constructed with 5UMH＿A of 83.5% homology as template showed that the enzyme was a monomer and Ramachandran plot showed that 93.5% of the primary amino-acid sequences in the modeled structure of the CatA were in most favored regions,6.2% in additional allowed regions,but 0.4% in generously allowed regions,which implied that the CatA had a reasonable three-dimensional structure.The molecular docking results showed that the active pocket residues of CatA consisted of Ile105、Gly107、Pro108、Leu109、Tyr164、Tyr200、Arg221、His224、His226、Phe253.It is predicted that the catalytic ternary of Tyr164-His224-His226 participate in the ring-opening reaction of catechol based on the above results.The binding and thermodynamic parameters obtained from fluorescence spectra indicated that catechol could effectively quench the intrinsic fluorescence of CatA via static and dynamic quenching mechanisms and spontaneously formed CatA/catechol complex by the hydrophobic interactions.The results of UV–vis spectra,synchronous fluorescence,and CD spectra revealed obvious changes in the microenvironment and conformation of CatA,especially for the secondary structure.Finally,the effect of vital residues on catechol catalyzed by CatA was verified by site directed mutation technology based on the prediction results of LIGPLOT and the results showed that Tyr164-His224His226 significantly affected the catalytic efficiency of CatA on catechol,and Tyr164,His224 and His226 may be the key amino acid residues for CatA catalyzed ring-opening of catechol.The potential interaction of CatA-catalyzed catechol elucidated in this study deepens the understanding of the PAHs degradation mechanism and provides a theoretical basis for the subsequent improvement of PAHs biodegradation.